PROJECT III (Kl: KAISER) G protein-coupled receptors (GPCRs) are the largest family of transmembrane proteins in vertebrates and are the molecular targets for nearly half of the therapeutic drugs that are prescribed worldwide. The approximately 1,000 members of the GPCR family exhibit a conserved 7-transmembrane domain topology and mediate physiological responses to diverse ligands, such as hormones, neurotransmitters and sensory stimuli. The role of three ligand/receptor pairs, (1) KISS1/KISS1R; (2) PROK2/PROKR2; and (3) TAC3/TACR3, have been implicated in the neuroendocrine regulation of reproduction and have been identified as underlying reproductive disorders. However, there remains much to be learned about the domains of these receptors important to cell surface expression, ligand binding, and activation of cellular signaling pathways relevant to the function of these receptors in the control of GnRH release. The human mutations will serve as tools to learn about the biology of these GPCRs and their ligands, with implications for structure-function relationships of GPCRs. In our first aim we will build on our previous studies of mutations in KISS1 and KISS1R to further define the cellular mechanisms by which KISS1/KISS1R regulate GnRH. In the second aim, our studies will identify proteins (chaperones) that modulate intracellular trafficking of GPCRs involved in the neuroendocrine regulation of reproduction. These studies will focus initially on PROKR2 as a model, and our findings will be extended to studies of KISS1R and TACR3 to determine their general applicability. Finally, we propose to leverage human mutations to elucidate new features of GPCR structure-function relationships, focusing on poorly understood GPCR domains, recently identified and insufficiently studied receptors, and interactions between receptors.